The present invention relates to 6,9-disubstituted 2-[trans-(4-aminocyclohexyl)amino]-purines and methods of using the same for antineoplastic agents or for treatment for neuronal injury and degeneration.
Cell division, in both normal and neoplastic cells, is a tightly controlled event which occurs by defined stages. Quiescent cells which are not actively dividing, are in the G0 phase, as are those terminally differentiated or in a state of temporary arrest. The first phase is the first gap (G1) phase during which the cell prepares to synthesize DNA. In late G1 phase at what is termed a restriction point or R point, the cell commits to entering S phase during which DNA synthesis occurs. Upon completion of S phase, the cell enters the second gap (G2) phase during which the cell prepares to divide, which is followed by mitosis, or M phase.
Initial experiments in cell cycle regulation revealed the existence of a protein called xe2x80x9cMaturation Promoting Factorxe2x80x9d (MPF), a heterodimer with kinase activity. Later, comparison of subsequently identified proteins and their underlying genes revealed a family of yeast genes known as cell division control (cdc) genes. Further experiments demonstrated that some of the cdc genes encode kinases, and were later called cyclin-dependent kinases (cdks). As the result of this reclassification, some cell cycle proteins have dual designations, such as cdk1 which is also known as cdc2. The kinase component of the MPF is now identified as p34cdc2 and the regulatory subunit of MPF is now called cyclin B. Cyclins were first identified as proteins whose levels oscillated during the cell cycle and were specifically degraded at mitosis. To date, animal cyclins A-I and cdks 1-8 have been identified. To further complicate nomenclature, subtypes of cyclins and cdks have been identified, such as cyclins B1 and B2.
Subsequent research on cell regulation has demonstrated that the stages of cellular division are achieved in part by modulation cyclins and cyclin-dependent kinases (cdks). Cyclins sequentially regulate cdks and are characterized by a 100 amino acid homology region termed the xe2x80x9ccyclin boxxe2x80x9d which is involved in binding a protein kinase partner. Cdks are closely related in sequence and size (35-40 kDa) and are defined as protein kinases activated by bound cyclin regulatory subunits. Cdks contain a conserved active-site cleft of approximately 300 amino acids that is characteristic of all eukaryotic protein kinases. Thus, both the cyclins and cdks appear to be highly conserved protein families.
Isolation of individual cyclins and cdks has enabled further identification of the roles and interactions of each component in cell cycle phase transitions. Excess levels of cdks persist throughout the cell cycle. Activation of cdks occurs upon cyclin synthesis and binding to the catalytic cdk subunit, the result of which is stimulation of the cdk serine/threonine kinase activity. Complete cdk activation requires phosphorylation on a conserved threonine residue located in the T-loop by a cyclin-dependent kinase activating kinase (CAK), which is itself a cdk/cyclin complex composed of cyclin H and cdk7, and a third protein of about 32 kDa.
Inactivation of the cdk-cyclin complex can result from the phosphorylation of a threonine and/or tyrosine residue in the ATP-binding site of the cdk or from binding of one of a number of endogenous inhibitor proteins.
In G1 phase, D-type cyclins bind to several different cdks, including cdk2, cdk4, cdk5 and cdk6, but are most commonly associated with cdk4 and cdk6. D-type cyclins are thought to act as growth factor sensors, which link cell cycle progression to external cues. Cyclin E-cdk2 complexes appear in the mammalian cell cycle after the D-type cyclin-cdk complexes. Cyclin E synthesis is tightly regulated and occurs in late G1 and early S phase. The cyclin E-cdk2 complex is essential for the cell to begin DNA replication.
The G1 cyclins, cyclin D and cyclin E, are transiently produced proteins, with a half-life of about 20 minutes. The short half-life is thought to result from a PEST sequence in the C-terminal regions of these proteins, the degradation of which appears to be mediated by the ubiquitination pathway.
The G2 cyclins, cyclin A and cyclin B, are stable throughout interphase and specifically destroyed at mitosis through an ubiquitination pathway. Both cyclin A and cyclin B2 appear to be degraded only when complexed with their cdk partner [cyclinA-cdk2 and cyclin A/B-cdk1(cdc2)]. However, cyclin B1 destruction is connected with the integrity of the mitotic apparatus at the end of metaphase. If the spindle is incorrectly assembled, or chromosomes incorrectly aligned, then cyclin B1 destruction is prevented.
Retinoblastoma protein (Rb), a 105 kDa nuclear phosphoprotein, is a substrate of cyclin-cdk complexes of cdks-2, 4 and 6 in G1 phase and functions as one of the major checkpoint controls in the cell cycle via carefully orchestrated phosphorylation and dephosphorylation. In G0/G1, Rb exists in a hypophosphorylated state. As the cell progresses into late G1, Rb becomes hyperphosphorylated by D-cyclin complexes, which inactivates Rb and drives the cell into S phase resulting in cell cycle progression and cell division. This state of hyperphosphorylation of Rb remains in G2. During late M phase, Rb is dephosphorylated, thus returning to the hypophosphorylated state. Phosphorylation of the Rb protein alters its binding characteristics; in the hypophosphorylated state, Rb binds to and sequesters specific transcription factors, such as E2F, the binding of which prevents the exit from the G1 phase. Once cdks hyperphosphorylate Rb, the transcription factors are released which can then activate transcription of genes necessary for S phase progression, for example, thymdine kinase, myc, myb, dihydrofolate reductase, and DNA polymerase-xcex1.
Localization of cyclin-CDK complexes is also very suggestive about the role each complex plays in the pathway. Nuclear cyclins A and E bind to p107 and p130, possibly because they are in the nucleus. Mammalian cyclin B1 accumulates in the cytoplasm in G2 phase and translocates into the nucleus at the beginning of mitosis. Cyclin B associates with the spindle apparatus, in particular with the spindle caps, and it is thought that the cyclin B-cdc2 kinase may be involved in the formation of the spindle through phosphorylating components of the mitotic apparatus. In addition, cyclin B1 is part of a feedback mechanism ensuring correct assembly of the metaphase mitotic apparatus. Human cyclin B2 is almost exclusively associated with the membrane compartment, and in particular the Golgi apparatus. Cyclin B2-cdc2 is involved in the disassembly of the Golgi apparatus when cells enter mitosis.
Cdc2-cyclin B kinase is a key mitotic factor which appears to be highly conserved and is thought to be involved in cell cycle transitions in all eukaryotic cells. Histone H1 is a substrate for cdc2-cyclin B; histone H1 is selectively phosphorylated on specific sites in mitosis, which is thought to be important for chromatin condensation. The cdc2-cyclin B complex also phosphorylates lamin, which is responsible for nuclear lamina breakdown. The nuclear lamina is made up of a polymer of lamin subunits that are hyperphosphorylated at mitosis, and this phosphorylation is responsible for their disassembly. Lamins are part of the intermediate filament family of proteins, and cdc2-cyclin B phosphorylates a subset of the sites phosphorylated at mitosis on the cytoplasmic intermediate filament subunits, vimentin and desmin. Thus, the cdc2-cyclin B complex is involved in the reorganization of the cell architecture at mitosis.
In addition, cdc2-cyclin B is involved in the reorganization of microfilaments, through phosphorylation of non-muscle caldesmon, an 83 kDa protein that binds to actin and calmodulin, and inhibits actomyosin ATPase activity. At mitosis, caldesmon is phosphorylated by cdc2-cyclin B, which weakens its affinity for actin and causes it to dissociate from microfilaments.
Cdc2-cyclin B is implicated in actomyosin filament regulation, by phosphorylating the myosin in the contractile ring, which divides the cell into two (cytokinesis). In metaphase, the myosin II regulatory light chain (MLC) is phosphorylated on two main sites at the N-terminus. Once phosphorylated, the myosin is prevented from interacting with actin. At anaphase, these two sites are dephosphorylated.
Cdc2-cyclin B also plays a role in reorganization of the membrane compartment at mitosis. For example, cdc2-cyclin B phosphorylates rab1Ap and rab4p. When rab4p is phophorylated by cdc2-cyclin B, it dissociates from the membrane compartment.
At mitosis, most forms of transcription are inhibited. Again, cdc2-cyclin B plays a role in inhibition of pol III-mediated transcription by phosphorylating TFIIIB. Given that pol I, pol II and pol III-mediated transcription share several common factors, such as TATA-binding protein (TBA), it is likely that cdc2-cyclin B is involved in down-regulating all forms of transcription at mitosis.
Given the importance of cyclin/cdk complexes in triggering cell cycle division, they are under tight regulatory mechanisms. Since their initial discovery, cyclins and cdks have been shown to interact with other transcription factors and proteins involved in a broad range of cellular pathways. Cdk7 has been identified as a component in transcription factor IIH (TFIIH), which contains the RNA polymerase II C-terminal domain (CTD) kinase activity. More recently, cdk8 which partners with cyclin C, has also been discovered to phosphorylate the CTD of RNA polymerase II, but does not appear to possess CAK activity. Thus, it is clear that cdks participate in a broad range of cellular functions in addition to cell cycle regulation. CDK-inhibitor proteins (CDIs) are small proteins that bind and inactivate specific cyclin-CDK complexes, or monmeric CDKs. These inhibitors can be grouped into two families based on sequence and functional similarities. The INK4 family includes p15INK4B, p16INK4, p18 and p19 which specifically bind cdk4 and cdk6. p16INK4 and p15INK4B contain four ankyrin repeats and, in addition to sharing significant homology, are encoded by adjacent genes on the 9p12 locus.
High cellular levels of p16 results in inactivation of cdk4 because p16 binds cyclinD-cdk4 and cyclin D-cdk6 complexes. The gene for p16INK4 (MTS1) is recognized as a potential tumor suppressor gene, as it is rearranged, deleted or mutated in a large number of tumor cell lines, and in some primary tumors. In one study of hereditary melanoma, about half the families had germline mutations in the p16INK4 gene. Rb is a repressor of p16INK4. Inactivation of cellular Rb, either by mutation or viral antigens, correlates with increased levels of p16INK4. P16INK4, p15INK4B, and p18 inhibit binding of cyclin D with cdk4 and cdk6.
The second family of CDIs is the Kip/Cip family which includes p21Cip1,WAF-1, p27Kip1 and p57Kip2 p27KIP1 is present in proliferating cells in a latent or masked form. Upon stimulation, p27KIP1 is unmasked and binds to and inhibits cyclin-CDK4/6 complexes. The Kip/Cip family proteins have strong homology in the N-terminus, the region that binds the cyclin-cdk complexes. The Kip/Cip family proteins preferentially bind to and inhibits cyclin-cdk complexes involved in the G1 and S phase complexes over those involved in the M phase.
P21 (also known as WAF1, Cip1 and Sdi1) is induced by p53 and forms a ternary complex with proliferating cell nuclear antigen (PCNA), a subunit of DNA polymerase xcex4 in several cyclin-CDK2 complexes, including cyclins A, D1 and E. P21WAF-1 expression in growing, quiescent and senescent cells correlates with a role as a negative regulator of S phase entry. P21WAF-1 mRNA is upregulated as cells become senescent or quiescent, and after serum stimulation of quiescent cells, and decreases as cells enter S phase. p21 inactivates cyclin E-cdk2, cyclin A-cdk2, and cyclins D1-, D2- and D3-cdk4 complexes.
Genetic analysis of numerous human tumors reveals a disproportionate numer of altered cell cycle proteins, and it is this aberration that is thought to cause abnormal cell cycle. For example, cyclin D1 is the bcl-1/PRAD1 proto-oncogene that is either overexpressed or deregulated in a variety of human tumors. The cyclin D1/CCND1 gene, located at chromosome 11q13, is amplified in a number of cancers, mainly breast and non-small cell lung carcinomas. This correlates with the observation that overexpression of cyclin D1 is a common feature in the tumors with this specific 11q13 amplicon. The gene for p16 is rearranged, deleted or mutated in a large number of tumour cell lines, and in some primary tumours. Mutations in cdk4, specifically an Arg24Cys mutation, has been identified in two unrelated hereditary melanoma families. This mutation was found in 11/11 of the melanoma patients, 2/17 unaffecteds and 0/5 spouses (Zuo, L., et al., Nature Genetics 12 1996:97-99). This mutation has a specific effect on the p16INK4a binding domain of cdk4, but has no affect on the ability to bind to cyclin D and form a functional kinase. As a result of this mutation, the cyclin D/cdk4 complex is resistant to normal physiological inhibition by p16INK4a. Other studies have demonstrated that about half the familial melanoma kindreds show evidence of linkage to the region of chromosome 9p21 that contains the p16INK4a gene. The types of p16INK4a mutations identified include a nonsense mutation, splice donor mutation, an unidentified mutation that prevents p16INK4a transcription, and 3 missense mutants that are unable to bind to cdk4 or cdk6. Overexpression of cdk4 as a result of gene amplification has been identified in a study of 32 glioma cell lines (He, J., et al., Cancer Res. 54:5804-5807, 1994). This alteration was observed among the ten cases having intact p16 genes. Genetic analysis of glioma cell lines revealed that 24 of 32 glioma cell lines had one of two alternative genetic alterations, each of which indicates that increased cdk4 kinase activity is important to glial tumor development. Cdk4 maps to the long arm of chromosome 12 and is found overexpressed in certain tumors because of its amplification as a component of an amplicon that includes other relevant genes, such as SAS and MDM2. All of the above conditions lead to activation of cdk4. Overexpression of cyclins B1 and E in leukemic and solid tumor cell lines, as well as altered patterns of cyclin E expression in breast cancer has also been reported.
Cellular hyperproliferation occurs in a number of disease states. The most common hyperproliferative diseases are neoplasms, which are typically named according to the original source of the hyperproliferative tissue. Neoplasms are defined as new growths of animal or plant tissue that resemble more or less the tissue from which it arises, but serve no physiologic function, and are benign, potentially malignant or malignant in character. Neoplasms arise as the result of loss of normal controls, leading to unregulated growth. Neoplastic cells may lack differentiation and acquire the ability to invade local tissues and metastasize. Neoplasms may develop in any type of tissue of any organ at any age. The incidence, and mortality rate, of neoplasms generally increases with age, with certain neoplasms having peak incidence between the ages of 60 and 80 (e.g. prostate, stomach and colon). However, other neoplasms have a peak incidence from birth to 10 years of age (e.g. acute lymphoblastic leukemia). Diet, exposure to carcinogens, particularly use of tobacco, and familial predispositions also affect incidence of particular neoplasms.
Neoplastic cells differ from normal cells in a number of important aspects, including loss of differentiation, increased invasiveness and decreased drug sensitivity. Another important difference is the unchecked growth of cells, which is thought to result from loss of normal cellular control mechanisms of these cells are either deactivated, bypassed or otherwise disregarded, leaving the neoplastic cells to proliferate without regard to the normal controlling mechanisms. Neoplasm is an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissue, and persists in the same excessive manner after cessation of the stimuli which evoked the change.
Neoplasms are classified as either benign or malignant. Benign neoplasms exhibit slow, localized growth that is usually circumscribed due to their encapsulation by a fibrous connective tissue capsule. Whereas benign neoplasms rarely cause the death of the organism, untreated malignant neoplasms have a high probability of killing the organism. Malignant neoplasms are generally nonencapsulated, and usually exhibit a more rapid growth rate. Malignant neoplasms often invade surrounding tissues and vessel and spread to distant body sites. Malignant neoplasms are generically described as xe2x80x9ccancerxe2x80x9d or as xe2x80x9ctumorsxe2x80x9d; the latter term denotes swelling.
Myeloproliferative disorders are a group of disorders characterized by abnormal proliferation by one or more hematopoietic cell lines or connective tissue elements. Four disorders are normally included as myeloproliferative disorders: polycythemia vera (primary polycythemia; Vaquez"" Disease), myelofibrosis (agnogenic myeloid metaplasia), chronic myelogenous leukemia and primary (essential) thrombocythemia. Acute leukemia, especially erythroleukemia, and paroxysmal nocternal hemoglobinuria are also classified as myeloproliferative disorders. Each of these disorders is identified according to its predominant feature or site of proliferation. Although each results from proliferation of different cells, each has been shown to be caused by a clonal proliferation arising at the level of a pluripotent stem cell, which causes varying degrees of abnormal proliferation of erythroid, myeloid, and megakaryocytic precursors in the bone marrow. All myeloproliferative disorders have a tendency to terminate in acute leukemia.
Leukemias are malignant neoplasms of the blood-forming tissues. At least two viruses are associated with causing leukemias in humans. The Epstein-Barr virus is associated with Burkitt""s lymphoma and the human T-cell lymphotropic virus, also called human acute leukemia/lymphoma virus (HTLV-1) has been linked to some T cell leukemias and lymphomas. Exposure, especially prolonged exposure to chemical agents, such as benzene and some antineoplastics, or to ionizing radiation, genetic predisposition (e.g. Down""s syndrome) and some familial disorders (e.g. Fanconi""s anemia) result in predispositions to leukemias.
Development of leukemias appears to occur through a single cell cycle through two or more steps with subsequent proliferation and clonal expansion. Leukemias are currently classified according to their cellular maturity; acute leukemias are predominantly undifferentiated cell populations and chronic leukemias are more mature cell forms. Acute leukemias are further divided into lymphoblastic (ALL, also known as acute lymphocytic leukemia) and myeloid (AML, also known as acute myelocytic, myelogenous, myeloblastic, myelomonoblastic) types. They may be further classified by morphologic and cytochemical appearance according to the French-American-British (FAB) classification or according to type and degree of differentiation. Chronic leukemias are classified as either lymphocytic (CLL) or myelocytic (CML). CLL is characterized by the appearance of mature lymphocytes in the blood, bone marrow and lymphoid organs. CML is characterized by the predominance of granulocytic cells of all stages of differentiation in blood, bone marrow, liver, spleen and other organs.
Myelodysplastic Syndrome (MDS) is characterized as a clonal proliferative disorder in which a normal or hypercellular bone marrow is associated with anemia and dysmyelopoiesis. Hemapoietic cells which may proliferate include erythroid, myeloid and megakaryocytic forms. MDS is a relatively new designation of group of disorders known as Preleukemia, Refractory Anemias, Ph-Chromosome-Negative Chronic Myelocytic Leukemia, Chronic Myelomonocytic Leukemia and Agnogenic Myeloid Metaplasia. The FAB system provides further classification of Myelofibrosis.
Lymphomas are a heterogeneous group of neoplasms arising in the reticuloendothelial and lymphatic systems. The major types of lymphomas are Hodgkin""s disease and non-Hodgkin""s lymphoma, as well as the rarer Burkitt""s lymphoma and mycosis fungoides. Hodgkin""s disease is a chronic disease with lymphoreticular proliferation of unknown cause that may present in localized or disseminated form, and is further classified according to four histopathologic profiles. Non-Hodgkin""s lymphomas are a heterogeneous group of diseases consisting of neoplastic proliferation of lymphoid cells that usually disseminate throughout the body. The former terms, lymphosarcoma and reticulum cell sarcoma, are now being replaced with terms that reflect that cell of origin and biology of the disease. The Rappaport classification is based on the histopathology; on the degree of the differentiation of the tumor; and on whether the growth pattern is diffuse or nodular. The Lukes and Collins classification is based upon the cell of origin, specifically whether it is T cell or B cell derived, histiocytic (or monocytic) origin or unclassifiable. The International Panel Working Formulation of the National Cancer Institute categorizes non-Hodgkin""s lymphomas using the above classifications.
Burkitt""s lymphoma is a highly undifferentiated B cell lymphoma that tends to involve sites other than the lymph nodes and reticulendoethlial system. Burkitt""s lymphoma, unlike other lymphomas, has a specific geographic distribution, which suggests an unidentified insect vector and an infectious agent. Evidence points to the herpes like Epstein-Barr virus.
Mycosis fungoides is an uncommon chronic T cell lymphoma primarily affecting the skin and occasionally internal organs.
Plasma cell dyscrasias (PCDs), or monoclonal gammopathy, are disorders characterized by the disproportionate proliferation of one clone of cells normally engaged in immunoglobulin (Ig) synthesis, and the presence of a structurally and electrophoretically homogeneous IG or polypeptide subunit in serum or urine. The disorders may be primarily asymptomatic to progressive, overt neoplasms (e.g., multiple myeloma). The disorder results from disproportionate proliferation of one clone producing a specific Ig: IgG, IgM, IgA, IgD or IgE.
Multiple myeloma, also known as plasma cell myeloma or myelomatosis, is a progressive neoplastic disease characterized by marrow plasma cell tumors and overproduction of an intact monoclonal Ig (IgG, IgA, IgD or IgE) or Bence Jones protein, which is free monoclonal xcexa or xcex light chains. Diffuse osteoporosis or discrete osteolytic lesions arise due to replacement by expanding plasma cell tumors or a osteoclast-activating factor secreted by malignant plasma cells.
Macroglobulinemia, or primary or Waldenstrom""s macroglobulinemia, is a plasma cell dyscrasia involving B cells that normally synthesize and secrete IgM. Macrogolbulinemia is distinct from myeloma and other PCDs, and resembles a lymphomatous disease. Many patients have symptoms of hyperviscosity, fatigue, weakness, skin and mucosal bleeding and so forth.
Heavy chain diseases are neoplastic plasma cell dyscrasias characterized by the overproduction of homogenous xcex3, xcex1, xcexc, and xcex4 Ig heavy chains. These disorders result in incomplete monoclonal Igs. The clinical picture is more like lymphoma than multiple myeloma.
Hypersplenism is a syndrome in which circulating cytopenia is associated with splenomegaly. Treatment of patients with hypersplenism requires therapy for the underlying disease, not splenectomy. Lymphoproliferative and myeloproliferative diseases are some, but not the sole, causes of hypersplenism. Myeloproliferative disorders causing hypersplenism include polycythemia vera, myelofibrosis with myeloid metaplasia, chronic myelogenous leukemia and essential thrombocythemia. Chronic lymphocytic leukemia and the lymphomas (including Hodgkin""s disease) are specific lymphoproliferative disorders that may cause hypersplenism.
Lung tissue is the site for both benign and malignant primary tumors, as well as the site of metastasis from cancers of many other organs and tissues. Cigarette smoking causes an overwhelming percentage of lung cancers, estimated at over ninety percent of the cases in men and about seventy percent of the cases in women. Exposure to occupational agents such as asbestos, radiation, arsenic, chromates, nickel, chloromethyl ethers, poison gas, and coke oven emissions is also associated with lung cancer. The most common types of lung cancer are squamous cell, small and large cell and adenocarcinoma.
About ninety-five percent of the stomach cancers are carcinoma; less common are lymphomas and leiomyosarcomas. Gastric carcinomas are classified according to gross appearance; protruding, penetrating (the tumor has a sharp, well-circumscribed border and may be ulcerated) and spreading or miscellaneous, which has characteristics of two of the other types.
Pancreatic cancers may be exocrine tumors, which are mostly adenocarcinomas arising from duct cells rather than the acinar cells, or endocrine tumors, which include insulinoama. Gastrin-producing pancreatic tumors involving cells of the non-xcex2-type or in the duodenal wall can cause Zollinger-Ellison Syndrome, a syndrome marked by hypergastrinemeia. Sometimes other endocrine abnormalities, particularly with the parathyroids, or pituitary and adrenal glands cause a polyglandular disorder known as multiple endocring neoplasia (MEN). Non-xcex2 islet cell tumors may cause a syndrome known as Vipoma Syndrome, which is characterized by prolonged massive watery diarrhea.
Neoplasms of the bowel include tumors of the small intestine, tumors of the large intestine, and cancer of the colon and rectum. Benign small intestine tumors may arise from jejunal and ileal neoplasms, including leiomyomas, lipomas, neurofibromas, and fibromas. Malignant small intestine tumors, such as adenocarcinomas, are uncommon, and typically arise in the proximal jejunum. Patients with Crohn""s disease of the small intestine are more prone to such adenocarcinomas rather than patients with Crohn""s disease of the colon. In patients with Crohn""s disease, the tumors tend to occur distally in the bypassed or inflamed loops of the bowel. Carcinoid tumors typically arise in the small bowel, especially the ileum, and in about half the cases, multiple tumors exist. Kaposi""s sarcoma, which occurs frequently in transplant recipients and AIDS patients, have gastrointestinal involvement in about half the cases. Lesions may occur anywhere in the GI tract, but are usually found in the stomach, small intestine, or distal colon.
Tumors of the large bowel include polyps of the colon and rectum. Polyps are a mass of tissue that arises from the bowel wall and protrudes into the lumen. Polyps are classified on the basis of their histology, as tubular adenomas, tubulovillous adenomas, villous adenomas, hyperplastic polyps, hamartomas, juvenile polyps, polypoid carcinomas, pseudopolyps, lipomas, leiomyomas and even rarer tumors.
Malignant tumors may also arise in the anorectum. These are epidermoid (squamous cells) carcinoma of the anorectum which comprise about three to five percent of rectal and anal cancers.
In Western countries, cancer of the colon and rectum are second to lung cancer in accounting for more new cases each year. In the USA, about 75,000 people died of these cancers in 1989; about 70% occurred in the rectum and sigmoid colon, and 95% were adenocarcinomas.
Neoplasms of the liver include benign neoplasms, which are relatively common but often undetected, and malignant neoplasms. Hepatocellular adenoma is the most important benign liver neoplasm. Asymptomatic small hemangiomas occur in one to five percent of adults. Bile duct adenomas and other mesenchymal neoplasms also occur, but are relatively rare. Malignant neoplasms of the liver are the most common form of hepatic tumor, and the liver is a frequent site of bloodborne metastases, usually from lung, breast, colon, pancreas and stomach primary tumors. The incidence of hepatocellular carcinoma is linked with chronic hepatitis B virus in certain parts of Africa and Southeast Asia. In North America, Europe and other areas of low prevelence, most of the patients have underlying cirrhosis. Fibrolamellar carcinoma is a distant variant of hepatocellular carcinoma with characteristic morphology of malignant hepatocytes enmeshed in lamellar fibrous tissue. Fibrolamellar carcinoma usually affects relatively young adults, and has no association with preexisting cirrhosis, chronic hepatitis B virus infection or other known risk factors. Other primary malignancies of the liver include cholangiocarcinoma (a tumor arising from intrahepatic biliary epithelium), hepatoblastoma (which is one of the most common cancers in infants) and angiosarcoma (which is associated with industrial exposure to vinyl chloride). Leukemia and related disorders may involve hepatic tissues, thought to be the result of infiltration with abnormal cells.
Multiple Endocrine Neoplasia (MEN) Syndromes are a group of genetically distinct familial diseases involving adenomatous hyperplasia and malignant tumor formation in several endocrine glands. Three distinct syndromes have been identified. Type I (MEN-I) is characterized by tumors of the parathyroid glands, pancreatic islets, and the pituitary. Type II (MEN-II) is characterized by medullary carcinoma of the thyroid, pheochromocytoma and hperparthyroidism. Type III (MEN-III) is characterized by multiple mucosal neuromas, medullary carcinoma of the thyroid, and pheochromocytoma.
Carcinoid syndrome is usually caused by metastatic intestinal carcinoid tumors that secrete excessive amount of vasoactive substances, including serotonin, bradykinin, histamine, prostaglandins and polypeptide hormones. Abnormal levels of these subtances cause a variety of symptoms, often episodic cutanteous flushing, cyanosis, abdominal cramps, diarrhea, and valvular heart disease.
Neoplasms of the bone and joints may be benign or malignant. Benign tumors of the bone include osteochondromas (osteocartilaginous exostoses), which are the most common benign bone tumors in children between ages 10 to 20, benign chondromas (which are located within the bone), which occur most commonly in children and young adults between the ages 10 to 30, chondroblastoma (which arises in an epiphysis), which is rare, but most common in children between the ages of 10 to 20, chondromyxofibromas, osteoid osteoma, giant cell tumors and fibromatous lesions. Primary malignant tumors of the bone include osteogenic sarcoma (osteosarcoma), which is the second most common primary bone tumor, fibrosarcomas, malignant fibrous histiocytoma, chondrosarcomas, mesenchymal chondrosarcoma, Ewing""s tumor (Ewing""s sarcoma), malignant lymphoma of bone, multiple myeloma, and malignant giant cell tumor.
Primary cancers of other tissues may metastasize to bone tissue. The most common are carcinomas arising in the breast, lung, prostate, kidney, and thyroid.
Central nervous system (CNS) neoplasms are generally classified according to the organ. Primary intracranial neoplasms are subdivided into six classes: tumors of (1) the skull; (2) the meninges; (3) the cranial nerves; (4) the neuroglia and ependyma; (5) pituitary or pineal gland; and (6) those of congenital origin. Skull neoplasms include osteoma, hemangioma, granuloma, xanthoma, and osteitis deformans. The meninges neoplasms include meningioma, sarcoma, and glomatosis. The cranial nerve neoplasms include glioma of the optic nerve, and schwannoma of the 8th and 5th cranial nerves. The neuroglia neoplasms include gliomas and ependymomas. The pituitary or pineal body neoplasms include pituitary adenoma and pinealoma. The congenital origin neoplams include craniopharyngioma, chordoma, germinoma, teratoma, dermoid cyst, agioma and hemangioblastoma.
Spinal cord neoplasms are lesions that compress the spinal cord or its roots, arising from the cord parenchyma, roots, meninges, or vertebrae. Primary spinal cord neoplasms are much less common than intracranial tumors. Metastatic lesions are common and may arise from carcinomas of the lung, breast, prostate, kidney, thyroid or lymphoma.
Genitourinary neoplasms occur at any age and in both sexes; however, they account for about 30% of cancer in the male and 4% in the female. Adenocarcinoma of the prostate accounts for a significant number of malignancies in men over 50. Prostate adenocarcinoma is thought to be hormone related and its pathology is typically glandular. Carcinoma of the kidney, adenocarcinoma, is only about one to two percent of adult cancers, but most solid kidney tumors are malignant. Wilms"" tumors, an embryonal adenomyosarcoma of the kidneys, occurs fetally and is often not diagnosed for several years. Renal pelvis and ureter neoplasms are histologically similar. Urinary bladder neoplasms may be induced by known urinary carcinogens such as aniline dyes, and the most common is transitional cell carcinoma, less common is squamous cell carcinoma. Rarer genitourinary neoplasms include carcinoma of the urethra, and penis. Neoplasms of the testis account for the majority of solid malignancies in males under 30. Most malignant testicular tumors arise from the primordial germ cell and are classified according to the cell type involved.
Breast cancer is the most common cancer in women. In the USA, the cumulative risk for women of all ages of developing breast cancer is about 10%, but that of dying from the disease is only about 3.6%. However, the risk increases with age, a family history of breast cancer, exposure to radiation, and even diet is implicated in higher risk.
Breast cancers are routinely typed for estrogen- and progesterone-receptor analysis. About two thirds of the patients have estrogen-receptor positive (ER+) breast tumors. Tumors which are progesterone positive are thought to have functional estrogen receptor and the presence of both receptors gives a greater likelihood of favorable response to endocrine treatment than the presence of just one receptor. Endocrine therapy, usually tamoxifen, is preferred in estrogen receptor-positive tumors. Estrogens and androgens are also effective, but less favored due to undesirable side effects induced by higher levels of these hormones than other forms of endocrine treatment. Breast cancer may metastasize to almost any organ in the body, but most common sites of metastatisis are the lung, liver, bone, lymph nodes and skin.
Lobular carcinoma in situ (LCIS) or lobular neoplasia, is most frequently found in premenopausal women. Ductal carcinoma in situ (DCIS) occurs in both pre- and postmenopausal women. DCIS forms a palpable mass. LCIS and DCIS account for about 90% of all breast cancers. The rarer forms, medullary and tubular lesions, have a somewhat better prognosis.
The most common gynecologic neoplasms are endometrial carcinomas, which ranks fourth in frequency after breast, colorectal and lung cancers in women. Endometrial carcinomas are characterized by their clinical staging, ranging from in situ at stage 0, to metastasis to distant organs at stage IVB. Endometrial carcinomas typically produce estrogen and the current treatment approaches are surgery and progesterone therapy.
Ovarian cancers account for about 18% of all gynecologic neoplasms. About 80% of malignant ovarian cancers arise from the ovarian epithelium and are classified according to their histology. Tumors may also arise from germ cells or stroma.
Vulvar carcinoma accounts for about 3-4% of all gynecologic neoplasms. Vulvar carcinoma usually occurs after menopause, and about 90% are squamous cell carcinomas. About 4% are basal cell carcinomas and the rest include intraepithelial carcinomas, adnocarcinoma of Bartholin""s gland, fibrosarcoma and melanoma.
Vaginal carinoma accounts for about 1% of gynecologic malignancies, with a peak incidence from about ages 45 to 65. About 95% of vaginal carcinomas are squamous cell carcinoma. Primary carcinoma of the oviduct is rare, and typically spread directly or by the lymphatics.
Trophoblastic disease or neoplams of trophoblastic origin, can follow intra- or extrauterine pregnancy. A degenerating pregancy results in a hydatidiform mole of which about 80% are benign.
Neoplasms may arise in the ear canal and affect hearing. Ceruminomas also arise, are typically malignant despite appearing benign histologically and are treated by surgical removal. Basal cell and squamous cell carcinomas frequently develop on the external ear as the result from regular sun exposure, and are also typically treated by surgical removal. The middle ear may be the site of squamous cell carcinomas. Nonchromaffin paragangliomas may arise in the temporal bone.
The most common malignant tumor in the nose and paranasal sinuses is squamous cell carcinoma; less common are adenoid cystic and mucoepidermod carcinomas, malignant mixed tumors, adenocarcinomas, lymphomas, fibrosarcomas, osteosarcomas, chondrosarcomas, and melanomas.
Squamous cell carcinoma of the nasopharynx is more commonly observed in children and young adults.
The most common malignancies of the upper respiratory tract are squamous cell carcinomas of the tonsil and of the larynx. Both are more common in males and are associated with tobacco smoking and ethanol ingestion; about 85% of patients with cancer of the head or neck have a history of ethanol and tobacco consumption.
In the head and neck, about 90% of the cancers are squamous cell (epidermoid) carcinoma. Melanomas, lymphomas and sarcomas are relatively rare forms of primary head and neck cancers. Cancers of the head and neck are classified according to the size and site of involvement of the primary neoplasm; number and size of metastases to the cervical lymph nodes; and evidence of distant metastases.
Ophthalmologic cancers may arise in the skin of the eyelids and may be benign or neoplastic. Common benign growths are xanthelasmas, which form yellow-white flat plaques of lipid material subcutaneously. Basal cell carcinomas are more common; treatment is typically surgical removal or radiation therapy. Other less common malignant tumors are squamous cell or meibomian gland carcinomas and other types of melanomas. The most common primary ocular malignancy is malignant melanoma of the choroid.
Tumors also arise in the skin tissue, and include benign tumors such as moles, lipomas and the like, as well as malignant tumors. About 40-50% of malignant melanomas arise from melanocytes in moles. Malignant skin cancers are either basal cell or squamous cell carcinomas and frequently arise in sun-exposed areas of skin. They are the most common malignancies, and the incidence is rising. Less common malignancies include malignant melanoma, Paget""s disease of the nipple or estramammary Patent""s, Kaposi""s sarcoma (KS), and cutaneous T cell lymphoma (mycosis fungiodes). The incidence of KS is increasing as the result of the increased incidence of AIDS. KS arises in about one third of patients with AIDS.
Oral cancers account for about 5% of cancers in men and 2% of cancers in women. The most common form of oral cancer is squamous cell carcinoma. Incidence increases with age and risk factors, particularly tobacco and alcohol consumption.
Surgery is the oldest effective form of treatment of neoplasms. Success is largely achieved if the neoplasm is detected in its early stages and has not metastasized. Radiation is also important therapy, and is the favored therapy of many neoplasms such as Hodgkin""s disease, early stage non-Hodgkin""s lymphomas, and squamous cell carcinoma of the head and neck. Radiation has proven very successful as an adjunct to surgery and antineoplastic drugs.
Antineoplastic drugs are also useful in the treatment of neoplasms, and are classified according to their mechanism of action. Numerous combinations, typically of antineoplastic drugs with differing mechanisms of action, have proven to be particularly effective therapy, permit lower doses and frequently minimize negative side effects. Antineoplastic drugs frequently target fundamental biological processes necessary for cell replication or growth.
Alkylating agents, such as mechlorethamin and cyclophosphamide, alkylate DNA, and restrict DNA replication.
Antimetabolites, which are directed to disruption of necessary cell division pathways, include:
Folate antagonists bind to dehydrofolate reductase and interfere with pyrimidine synthesis. Folate antagonists are S-phase specific. Methotrexate is a very commonly used antineoplastic folate antagonist.
Purine antagonists block de novo purine synthesis and are S-phase specific. 6-Mercaptopurine is an example of a purine antagonist.
Pyrimidine antagonists interfere with thymidylate synthase to reduce thymidine production and are S-phase specific. A frequently used pyrimidine antagonist is 5-fluorouracil.
Cytarabine inhibits DNA polymerase and is S-phase specific.
Plant alkyloids include vincas, such as vinblastine and vincristine, and podophyllotoxins, such as etoposide. Plant alkyloids are effective in the metaphase and inhibit mitosis by a variety of mechanisms including altering microtubular proteins.
Antibiotics include doxorubicin and daunomycin, which intercalate between DNA strands to inhibit the uncoiling of DNA; bleomycin, which causes incisions in DNA strands; and mitomycin, which inhibits DNA synthesis by acting as a bifunctional alkylator.
Nitrosureas include carmustine and lomustine and alkylate DNA or cause carbamoylate amino acids in proteins.
Inorganic ions, such as cisplatin, cause inter- and intracalation of DNA strands to inhibit the uncoiling of DNA.
Biologic Response Modifiers, such as the interferons, have antiproliferative effects, but their specific role is not known. Interferons include xcex1 (leukocyte) interferon, xcex2 (fibroblast) interferon and xcex3 (lymphocyte) interferon.
Enzymes, such as asparaginase, are also used to alter metabolic pathways important in cancerous cells. Asparaginase depletes the cell of asparagine, on which leukemic cells depend.
Hormones and their analogs, such as tamoxifen, flutamide and progesterone, have non-specific effects but are useful to treat certain neoplams which are known to be hormone responsive, especially breast, ovarian and prostate neoplasms. Tamoxifen, frequently used in the treatment of breast neoplasms, places cells at rest, and binds to the estrogen receptor. Flutamide, frequently used in the treatment of prostate neoplasms, binds the androgen receptor.
Cytokinins are naturally occurring and artificial plant growth regulators. Natural cytokinins tend to be non-specific inhibitors of various protein kinases. The molecular mechanisms by which cytokinins regulate cell growth and division are still being determined. Studies have indicated that cytokinins may increase accessibility of the DNA template, activate RNA polymerases, affect polyadenylation and secondary structure of mRNA and stimulate formation and activity of polyribosomes. Cytokinins are thought to affect cell division by interacting with regulatory proteins of the cell cycle. Both cytokinins and cyclin-dependent kinases (cdks) act at multiple and similar control points of cell cycle, for example, at the G1/S and G2/M transitions and S and M phases.
Olomoucine, 6-(benzylamino)-2-[(2-hydroxyethyl)amino]-9-methylpurine, was first discovered as an herbicide. More recently, it has been discovered that Olomoucine is an artificial cytokinin, which specifically inhibit some cdks, including p34cdc2/cyclin B kinases, at micromolar concentration, but has no effect on other major protein kinases such as cAMP- and cGMP-dependent kinases, and protein kinase C. Olomoucine has recently been shown to have good selectivity for the CDK-cyclin protein kinases, but only has moderate inhibitory activity, with an IC50 of about 7 xcexcM. Vesely, J., et al., Eur. J. Biochem., 1994, 224, 771-786. A 2.4 A crystal structure of olomucine co-crystallized with cdk2 revealed that the purine portion of olomoucine binds in the conserved ATP binding pocket, while the benzylamino group extends into a region of the active site unique to the cdk2 kinases.
Roscovitine, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine, is a recently synthesized purine which has been shown to have selectivity towards some cyclin-dependent kinases and to be 10-fold more active on cdk2 and cdc2 than olomoucine (Meijer, L., et al., Eur. J. Biochem., 243:527-536, 1997 and PCT/FR96/01905). Meijer et al report that most kinases are not significantly inhibited by roscovitine. However, cdc 2-cyclin B, cdk 2-cyclin A, cdk 2-cyclin E and cdk 5-p35 are substantially inhibited with IC50 values of 0.65, 0.7, 0.7 and 0.2 xcexcM, respectively. In contrast, roscovitine displayed IC50 values of greater than 100 xcexcM for cdk 4-cyclin D1 and cdk 6-cyclin D2.
Havlicek, L., et al., J. Med. Chem. (1997)40:408-412 report that Roscovitine, and related analogs substituted in the 2, 6 and/or 9 positions, inhibit p34cdc2 cyclin B kinases. None of the analogs had superior IC50 values over the (R) enantiomer of Roscovitine, which had an IC50 value of 0.2 xcexcM. The (S) enantiomer had an IC50 value of 0.8 xcexcM; the racemic mixture (R/S) had an IC50 value of 0.65 xcexcM. These authors conclude that the N6-benzyl substituent of Roscovitine was superior over the isopentenyl or cyclohexylmethyl substituents.
The National Cancer Institute (NCI) is a US Government-run organization directed at the discovery and development of novel therapeutic oncology products. In 1985, the NCI established a new cancer screening strategy involving human tumor cell lines in an in vitro assay as the primary cancer screen. A total of sixty human tumor cell lines, derived from seven cancer types (lung, colon, melanoma, renal, ovarian, brain and leukemia) were selected for inclusion in the NCI panel (Grever, M. R., et al., Seminars in Oncology, 19:1992:622-638). The protocols used in the assays have also been reported in the literature. American Type Tissue Collection (ATCC) acts as a depository for these and other tumor cell lines. Useful human tumor cell lines include the following:
MCF7: human breast adenocarcinoma, hormone-dependent;
MDA-MB-23 1: human breast adenocarcinoma, hormone-independent;
HT-29: human colon adenocarcinoma, moderately well-differentiated grade II;
HCT-15: human colon adenocarcinoma;
A549: human non-small cell lung carcinoma;
DMS-1 14: human small cell lung carcinoma;
PC-3: human prostate adenocarcinoma, hormone-independent; and
DU 145: human prostate carcinoma, hormone-independent.
Skehan, P., et al., J. Natl. Cancer Inst. 82: 1107-1112, 1990 sets forth useful protocols for using such tumor cell lines for screening antineoplastic drugs.
Meijer, et al., supra, report that roscovitine inhibits the proliferation of the NCI disease-oriented in vitro screen, i.e., 60 human tumour cell lines comprising nine tumour types (leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, breast cancer) with an average IC50 value of 16 xcexcM. The results of individual tumour lines were not reported.
Two distinct cdk inhibitors, flavopiridol and olomoucine, suppress the death of neuronal PC12 cells and sympathetic neurons in two model systems of neuronal survival (Park et al., J. Biol. Chem. 271(14):8161-8169, 1996). The concentration of each required to promote survival correlated with the amount required to inhibit proliferation. Neuronal apoptosis is an important aspect of both nervous system development and a component of neuronal injury and disease.
The PC12 cell line was initially derived from a rat adrenal medullary pheochromocytoma. When grown in serum-containing medium, PC12 cells divide and resemble precursors of adrenal chromaffin cells and sympathetic neurons. Upon addition of nerve growth factor (NGF), PC12 cells attain the phenotypic properties of sympathetic neurons. Upon removal of either serum or serum and NGF, both naive and neuronally differentiated PC12 cells undergo apoptosis, which is analogous to the response of sympathetic neurons.
The role of cell cycle regulation in apoptosis may be demonstrated by withdrawal of NGF or serum which results in uncoordinated cell cycle progression and cell death from naive PC-12 cells. Cdk inhibitors did not prevent the death of these proliferation competent naive PC-12 cells after removal of trophic support. Post-mitotic differentiated or sympathetic neurons are hypothesized to attempt inappropriate re-entry of the cell cycle following withdrawal of NGF which results in cell death. However, exposure to flavopiridol or olomoucine which inhibit cdks prevented apoptosis in these cells.
Changes in the activity of cdks and cyclins are observed during apoptosis of many different cell types. Camptothecin- or araC-induced apoptosis of HL60 cells is associated with elevated cdc2 activity and cyclin E-associated kinase activity. Camptothecin-induced apoptosis of RKO cells is associated with an increase in expression of cyclin D1.
Camptothecin causes apoptotic death of rat cerebral cortical neurons. Morris and Geller, J. Cell Biol. 134:757-770(1996). Camptothecin-treated nonproliferating neuronally differentiated PC12 cells die within 6 days after treatment, and cultured rat sympathetic neurons die within 5 days after treatment, even in the presence of NGF. Park et al., J. Neurosci. 17(4):1256-1270(1997). However, administration of either both, or individual olomoucine or flavopiridol, in the presence or absence of camptothecin resulted in approximately 30% cell death at day 6. Maximal protection of PC12 cells, or rat sympathetic neurons, from death was observed with 1 xcexcM flavopiridol and 200 xcexcM olomoucine, which are the minimum concentrations that fully inhibit DNA synthesis by proliferating PC12 cells. Administration of iso-olomoucine, an inactive analog of olomoucine, failed to prevent the cell death of camptothecin-treated neuronal cells
Flavopiridol and olomoucine were also shown to protect against camptothecin-induced cortical neuronal death. Park et al., J. Neurosci. 17(4):1256-1270(1997). The IC50 values of flavopiridol and olomoucine were 0.1 xcexcM and 100 xcexcM, respectively. Administration of iso-olomoucine failed to prevent the cell death of camptothecin-treated neuronal cells.
There are several implications of the above observations. It is well recognized that patients treated with radiation or antineoplastic agents experience undesirable side effects, including developing new neoplasms or undesirable cellular apoptosis. For example, some patients treated with high-dose araC for refractory leukemia develop a cerebellar toxicity syndrome, characterized by loss of Purkinje neurons. Winkelman and Hinges, Ann Neurol. 14:520-527(1983) and Vogel and Horouipian, Cancer 71:1303-1308(1993). Patients treated with cis-platinum have been reported to develop periperal neuropathies. Wallach, et al., J. Fla. Med. Assoc. 79:821-822(1992) and Mansfield and Castillo, AJNR Am. J. Neuroradiol. 15:1178-1180(1994). In view of these observations, either co-administration or sole administration of the present compounds in the treatment of neoplasms would reduce or preclude cellular apoptosis, in particular, neuronal damage caused by treatment with antineoplastic agents or radiation.
Cerebrovascular disease is the most common cause of neurologic disability in Western countries. The major specific types of cerebrovascular disease are cerebral insufficiency due to transient disturbances of blood flow, infarction, hemmorrhage, and arteriovenous malformation. Stroke generally denotes ischemic lesions. Undesirable neuronal apoptosis occurs in cerebrovascular disease. Treatment with inhibitors of cdks may be an approach to prevent is neuronal injury and degeneration in such cases.
The present invention provides novel compounds of the formula (I) 
wherein R is selected from the group consisting of R2, R2NH-, or R3R4Nxe2x80x94R5- wherein
R2 is selected from the group consisting of C9-C12 alkyl, 
wherein each R6 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)m-phenyl, wherein m is an integer 0-8; x is an integer 1-8; n is an integer 0-8; Z is selected from the group consisting of phenyl, heterocycle, cycloalkyl, and naphthanlene; and M is selected from the group consisting of hydrogen, C1-C4 alkyl, 
wherein each R6xe2x80x2 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)m-phenyl, wherein mxe2x80x2 is an integer 0-8; nxe2x80x2 is an integer 0-8; xxe2x80x2 is an integer 1-8; Q is hydrogen or C1-C4 alkyl; and Zxe2x80x2 is selected from the group consisting of phenyl, heterocycle, cycloalkyl, and napthalene; and
wherein each C9-C12 alkyl or Z is optionally substituted with 1 to 3 substituents, which may be the same or different, and which are selected from the group consisting of D, E, 
wherein each D is independently selected from the group consisting of trifluoromethyl, trifluoromethoxy, and C1-C4 alkoxy; each E is independently selected from the group consisting of Hal, OH, and C1-C8 alkyl; b is an integer 0-2; Zxe2x80x3 is selected from the group consisting of phenyl, heterocycle, cycloalkyl, and naphthalene; each R6xe2x80x3 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)mxe2x80x3-phenyl, wherein mxe2x80x3 is an integer 0-8; nxe2x80x3 is an integer 0-8; xxe2x80x3 is an integer 1-8; and Mxe2x80x2 is selected from the group consisting of hydrogen, C1-C4 alkyl, 
wherein each R6xe2x80x2xe2x80x3 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)mxe2x80x2xe2x80x3-phenyl, wherein mxe2x80x2xe2x80x3 is an integer 0-8; nxe2x80x2xe2x80x3 is an integer 0-8; xxe2x80x2xe2x80x3 is an integer 1-8; Qxe2x80x2 is hydrogen or C1-C4 alkyl; and Zxe2x80x2xe2x80x3 is selected from the group consisting of phenyl, heterocycle, cycloalkyl, and napthalene,
wherein the groups Mxe2x80x2 and Zxe2x80x3 may be optionally substituted with the groups Dxe2x80x2, Exe2x80x2 or 
wherein each R6xe2x80x3xe2x80x3 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)mxe2x80x3xe2x80x3-phenyl, wherein mxe2x80x3xe2x80x3 is an integer 0-8; xxe2x80x3xe2x80x3 is an integer 0-8; Qxe2x80x3 is hydrogen, C1-C4 alkyl or phenyl; each Dxe2x80x2 is independently selected from the group consisting of trifluoromethyl, trifluoromethoxy, and C1-C4 alkoxy; each Exe2x80x2 is independently selected from the group consisting of Hal, OH, and C1-C8 alkyl;
R3 and R4 are selected from the group consisting of hydrogen, C1-C4 alkyl and (CH2)y-phenyl, wherein y is an integer 0-8, with the proviso that R3 and R4 not both be hydrogen;
R5 is C1-C8 alkylene; and
R1 is selected from the group consisting of cyclopentyl, cyclopentenyl and isopropyl,
and the pharmaceutically acceptable salts, optical isomers, and hydrates thereof,
with the proviso that when R2 is the group 
wherein n is 1 or greater; R1 is isopropyl or cyclopentyl; R6 is hydrogen, C1-C4 alkyl, or (CH2)-phenyl; and Z is phenyl, heterocycle, or cycloalkyl, that Z is substituted with 1 to 3 substituents, which may be the same or different, and which are selected from the group consisting of 
wherein D, b, R6xe2x80x3, xxe2x80x3, nxe2x80x3, Mxe2x80x2, and Zxe2x80x3 are as previously defined.
In addition, the present invention provides a method of inhibiting cell cycle progression. More specifically, the present invention provides a method of inhibiting cdk-2.
The present invention also provides a method of preventing apoptosis in neuronal cells. A particularly preferred method of the present invention is preventing apoptosis of neuronal cells induced by antineoplastic agents or resulting from cerebrovascular disease. Another preferred embodiment of the present invention is the method of preventing apoptosis induced by oxygen depletion. A more preferred invention provides a method of preventing apoptosis induced cerebrovascular disease. Another preferred invention provides a method of preventing apoptosis induced by stroke or infarction.
The present invention provides a method of inhibiting the development of neoplasms. The present invention provides a method for treating a patient afflicted with a neoplastic disease state comprising administering a compound of the formula provided. It is preferred that the amount administered is a therapeutically effective amount of a compound of the formula. A preferred method of the present invention administers a single compound of the formula provided. Alternatively, a preferred method of the present invention administers an amount of a compound of the formula in conjunction with other antineoplastic agents.
In addition, the present invention provides a composition comprising an assayable amount of a compound of Formula (I) in admixture or otherwise in association with an inert carrier. The present invention also provides a pharmaceutical composition comprising an effective inhibitory amount of a compound of Formula (I) in admixture or otherwise in association with one or more pharmaceutically acceptable carriers or excipients.
The present invention provides novel compounds of the formula (I) 
wherein R is selected from the group consisting of R2, R2NH-, or R3R4Nxe2x80x94R5- wherein
R2 is selected from the group consisting of C9-C12 alkyl, 
wherein each R6 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)m-phenyl, wherein m is an integer 0-8; x is an integer 1-8; n is an integer 0-8; Z is selected from the group consisting of phenyl, heterocycle, cycloalkyl, and naphthanlene; and M is selected from the group consisting of hydrogen, C1-C4 alkyl, 
wherein each R6xe2x80x2 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)m-phenyl, wherein mxe2x80x2 is an integer 0-8; nxe2x80x2 is an integer 0-8; xxe2x80x2 is an integer 1-8; Q is hydrogen or C1-C4 alkyl; and Zxe2x80x2 is selected from the group consisting of phenyl, heterocycle, cycloalkyl, and napthalene; and
wherein each C9-C12 alkyl or Z is optionally substituted with 1 to 3 substituents, which may be the same or different, and which are selected from the group consisting of D, E, 
wherein each D is independently selected from the group consisting of trifluoromethyl, trifluoromethoxy, and C1-C4 alkoxy; each E is independently selected from the group consisting of Hal, OH, and C1-C8 alkyl; b is an integer 0-2; Zxe2x80x3 is selected from the group consisting of phenyl, heterocycle, cycloalkyl, and naphthalene; each R6xe2x80x3 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)mxe2x80x3-phenyl, wherein mxe2x80x3 is an integer 0-8; nxe2x80x3 is an integer 0-8; xxe2x80x3 is an integer 1-8; and Mxe2x80x2 is selected from the group consisting of hydrogen, C1-C4 alkyl, 
wherein each R6xe2x80x2xe2x80x3 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)mxe2x80x2xe2x80x3-phenyl, wherein mxe2x80x2xe2x80x3 is an integer 0-8; nxe2x80x2xe2x80x3 is an integer 0-8; xxe2x80x2xe2x80x3 is an integer 1-8; Qxe2x80x2 is hydrogen or C1-C4 alkyl; and Zxe2x80x2xe2x80x3 is selected from the group consisting of phenyl, heterocycle, cycloalkyl, and napthalene,
wherein the groups Mxe2x80x2 and Zxe2x80x3 may be optionally substituted with the groups Dxe2x80x2, Exe2x80x2 or 
wherein each R6xe2x80x3xe2x80x3 is independently selected from the group consisting of hydrogen, C3-C8 cycloalkyl, C1-C4 alkyl, and (CH2)mxe2x80x3xe2x80x3-phenyl, wherein mxe2x80x3xe2x80x3 is an integer 0-8; xxe2x80x3xe2x80x3 is an integer 0-8; Qxe2x80x3 is hydrogen, C1-C4 alkyl or phenyl; each Dxe2x80x2 is independently selected from the group consisting of trifluoromethyl, trifluoromethoxy, and C1-C4 alkoxy; each Exe2x80x2 is independently selected from the group consisting of Hal, OH, and C1-C8 alkyl;
R3 and R4 are selected from the group consisting of hydrogen, C1-C4 alkyl and (CH2)y-phenyl, wherein y is an integer 0-8, with the proviso that R3 and R4 not both be hydrogen;
R5 is C1-C8 alkylene; and
R1 is selected from the group consisting of cyclopentyl, cyclopentenyl and isopropyl,
and the pharmaceutically acceptable salts, optical isomers, and hydrates thereof,
with the proviso that when R2 is the group 
wherein n is 1 or greater; R1 is isopropyl or cyclopentyl; R6 is hydrogen, C1-C4 alkyl, or (CH2)m-phenyl; and Z is phenyl, heterocycle, or cycloalkyl, that Z is substituted with 1 to 3 substituents, which may be the same or different, and which are selected from the group consisting of 
wherein D,b,R6xe2x80x3,xxe2x80x3,nxe2x80x3,Mxe2x80x2, and Zxe2x80x3 are as previously defined.
As used herein, the term xe2x80x9cheterocyclexe2x80x9d means any closed-ring moiety in which one or more of the atoms of the ring are an element other than carbon and includes, but is not limited to the following: piperidinyl, pyridinyl, isoxazolyl, tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperazinyl, benzimidazolyl, thiazolyl, thiophene, furanyl, indolyl, 1,3-benzodioxolyl, tetrahydropyranyl, imidazolyl, tetrahydrothiophene, pyranyl, dioxanyl, pyrrolyl, pyrimidinyl, pyrazinyl, triazinyl, oxazolyl, purinyl, quinolinyl, and isoquinolinyl.
As used herein, the term xe2x80x9cC1-C4 alkylxe2x80x9d refers to a saturated or unsaturated, straight of branched chain hydrocarbyl radical of from one to four carbon atoms and includes, but is not limited to the following: methyl, ethyl, propyl, isopropyl, 1-propenyl, 2-propenyl, n-butyl, isobutyl, tertiary butyl, sec-butyl, 1-butenyl, 2-butenyl, 3-butenyl, and the like.
As used herein, the term xe2x80x9cC1-C8 alkylxe2x80x9d refers to a saturated or unsaturated, straight or branched chain hydrocarbyl radical of from one to eight carbon atoms and includes, but is not limited to the following: methyl, ethyl, propyl, isopropyl, 1-propenyl, 2-propenyl, n-butyl, isobutyl, tertiary butyl, sec-butyl, 1-butenyl, 2-butenyl, 3-butenyl, pentyl, neopentyl, hexyl, heptyl, octyl, and the like.
As used herein, the term xe2x80x9cC9-C12 alkylxe2x80x9d refers to a saturated or unsaturated, straight or branched chain hydrocarbyl radical of from nine to twelve carbon atoms and includes, but is not limited to the following: nonyl, decyl, undecyl, and dodecyl, and the like.
As used herein, the term xe2x80x9cC1-C8 alkylenexe2x80x9d refers to a saturated or unsaturated, straight of branched chain hydrocarbylene radical of from one to eight carbon atoms and includes, but is not limited to the following: methylene, ethylene, propylene, isopropylene, 1-propenylene, 2-propenylene, n-butylene, isobutylene, tertiary butylene, sec-butylene, 1-butenylene, 2-butenylene, 3-butenylene, pentylene, neopentylene, hexylene, heptylene, octylene, and the like.
As used herein, the term xe2x80x9ccycloalkylxe2x80x9d refers to a saturated or unsaturated alicyclic moiety containing three to eight carbon atoms and includes, but is not limited to, the following: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
As used herein, the designation 
refers to a sulfur atom which is optionally oxidized to a sulfoxide (b=1) or a sulfone (b=2).
As used herein, the term xe2x80x9cHalxe2x80x9d refers to a halogen moiety and includes fluoro, chloro, bromo, and iodo moieties.
As used herein, the term xe2x80x9coptical isomerxe2x80x9d or xe2x80x9coptical isomersxe2x80x9d refers to any of the various stereo isomeric configurations which may exists for a given compounds of Formula (I).
As used herein, the term xe2x80x9chydratexe2x80x9d or xe2x80x9chydratesxe2x80x9d refers to the reaction product of one or more molecules of water with a compound of formula (I) in which the Hxe2x80x94OH bond is not split and includes monohydrates as well as multihydrates.
As used herein, the term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to the reaction product of one or more molecules of any non-toxic, organic or inorganic acid with the compounds of Formula (I). Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include mono, di and tricarboxylic acids. Illustrative of such acids are, for example, acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salacylic acid, 2-phenoxybenzoic acid and sulfonic acids such as methane sulfonic acid, trifluoromethane sulfonic acid and 2-hydroxyethane sulfonic acid.
The compounds of Formula (I) can be prepared by utilizing procedures and techniques well known and appreciated by one of ordinary skill in the art. A general synthetic scheme for preparing these compounds is set forth in Scheme A wherein all substituents, unless otherwise indicated, are as previously defined. 
In Scheme A, step a, 2,6-dichloropurine (1) is reacted with the appropriate alcohol of structure 2 to give the corresponding 9-substituted-2,6-dichloropurine compound of structure 3 using techniques and procedures well known to one of ordinary skill in the art.
For example, 2,6-dichloropurine (1) can be reacted with the appropriate alcohol of structure 2 in the presence of triphenylphosphine and diethyl azodicarboxylate in a suitable anhydrous aprotic solvent, such as tetrahydrofuran. The reactants are typically stirred together at room temperature for a period of time ranging from 5 hours to 5 days. The resulting 9-substituted-2,6-dichloropurine of structure 3 may be recovered from the reaction zone by extractive methods as is known in the art or more typically, the resulting 9-substituted-2,6-dichloropurine of structure 3 is recovered by removal of solvent following by charging directly onto a silica gel column and eluting with a sutiable solvent, such as methylene chloride or mixture of solvents, such as a mixture of hexane and ethyl acetate. The crude 9-substituted-2,6-dichloropurine of structure 3 may then be purified by chromatography or may be used in the next step without purification.
In step b, the 6-chloro functionality of the 9-substituted-2,6-dichloropurine of structure 3 is reacted with an appropriate amine of structure 4 to give the corresponding 9-substituted-6-amino-2-chloropurine compound of structure 5.
For example, the 9-substituted-2,6-dichloropurine of structure 3 can be reacted with the appropriate amine of structure 4 in a suitable anhydrous polar solvent such as ethanol. The reactants are typically stirred together at reflux temperatures for a period of time ranging from 30 minutes to 3 days. The resulting 9-substituted-6-amino-2-chloropurine of structure 5 is recovered from the reaction zone by extractive methods as are known in the art, or, if the 9-substituted-6-amino-2-chloropurine of structure 5 precipitates out of solution, it may be recovered by filtration.
In step c, the 2-chloro functionality of the 9-substituted-6-amino-2-chloropurine of structure 5 is reacted with 1,4-cyclohexanediamine (6) to give the corresponding compound of Formula I.
For example, the appropriate 9-substituted-6-amino-2-chloropurine of structure 5 can be reacted with a molar excess of 1,4-cyclohexanediamine (6). The reactants are typically placed in a pressure tube, sealed, and heated at a temperature of from about 80xc2x0 C. to about 150xc2x0 C. for a period of time ranging from 30 minutes to 3 days. The resulting compound of Formula I is recovered from the reaction zone by extractive methods as are known in the art and may be purified by chromatography.
Starting materials for use in the general synthetic procedures outlined in Scheme A are readily available to one of ordinary skill in the art. For example, certain 4-aminopiperidines and 3-aminopyrrolidines of structure 4 may be prepared as described in Schemes B and C below.
Starting amines of structure 4 for use in Scheme A which are 4-amino-1-piperidine and 3-amino-1-pyrrolidine derivatives (structure 4xe2x80x2) may be prepared as shown in Scheme B, wherein all substituents, unless otherwise indicated, are as previously defined. 
In Scheme B, step a, the free amino functionality of an appropriate 4-carboxamide-1-piperidine or 3-carboxamide-1-pyrrolidine derivative of structure 7 is reacted with the appropriate alkyl halide of structure 8 to give the corresponding 4-carboxamide-1-alkylated-piperidine or 3-carboxamide-1-alkylated-pyrrolidine of structure 9.
For example, the 4-carboxamide-1-piperidine or 3-carboxamide-1-pyrrolidine of structure 7 can be reacted with the appropriate alkyl halide of structure 8 in a suitable aprotic organic solvent, such as 3-pentanone, in the presence of a suitable base, such as cesium carbonate, and a catalytic amount of a suitable alkylation catalyst, such as potassium iodide. The reactants are typically stirred together at reflux temperature for a period of time ranging from 30 minutes to 12 hours. The resulting 4-carboxamide- -alkylated-piperidine or 3-carboxamide-1-alkylated pyrrolidine of structure 9 is recovered from the reaction zone by filtration and evaporation of solvent
In step b, the carboxamide functionality of the appropriate 4-carboxamide-1-alkylated piperidine or 3-carboxamide-1-alkylated pyrrolidine of structure 9 is dehydrogenated to give the corresponding 4-amino-1-alkylated-piperidine or 3-amino-1- alkylated-pyrrolidine of structure 4xe2x80x2.
For example, the appropriate 4-carboxamide-1-alkylated piperidine or 3-carboxamide-1-alkylated pyrrolidine of structure 9 is reacted with a molar excess of bis(trifluoroacetoxy)-iodobenzene in a suitable aprotic polar solvent such as acetonitrile. The reactants are typically stirred together at a temperature of about 50xc2x0 C. to about 95xc2x0 C. for a period of time ranging from 30 minutes to 5 hours. The resulting 4-amino-1-alkylated-piperidine or 3-amino-1-alkylated-pyrrolidine of structure 4xe2x80x2 is recovered from the reaction zone by extractive methods as are known in the art.
Alternatively, starting amines of structure 4 for use in Scheme A which are 4-amino-1-piperidine and 3-amino-1-pyrrolidine derivatives (structure 4xe2x80x2) may be prepared as shown in Scheme C, wherein all substituents, unless otherwise indicated, are as previously defined. 
Scheme C, step a, the free amino functionality of an appropriate 4-piperidone or 3-pyrrolidone derivative of structure 10 is reacted with the appropriate alkyl halide of structure 8 to give the corresponding 1-alkylated-4-piperidone or 1-alkylated-3-pyrrolidone of structure 11.
For example, the 4-piperidone or 3-pyrrolidone of structure 10 can be reacted with the appropriate alkyl halide of structure 8 in a suitable aprotic organic solvent, such as 3-pentanone, in the presence of a suitable base, such as cesium carbonate, and a catalytic amount of a suitable alkylation catalyst, such as potassium iodide. The reactants are typically stirred together at reflux temperature for a period of time ranging from 30 minutes to 12 hours. The resulting 1-alkylated-4-piperidone or 1-alkylated-3-pyrrolidone of structure 11 is recovered from the reaction zone by filtration and evaporation of solvent.
In step b, the ketone functionality of the appropriate 1-alkylated-4-piperidone or 1-alkylated-3-pyrrolidone of structure 11 is reacted with hydroxylamine hydrochloride (12) to give the corresponding 1-alkylated-4-piperidone oxime or 1-alkylated-3-pyrrolidone oxime of structure 13.
For example, the 1-alkylated-4-piperidone or 1-alkylated-3-pyrrolidone of structure 11 is reacted with hydroxylamine hydrochloride (12) in the presence of a suitable base, such as sodium acetate in a suitable protic solvent, such as aqueous ethanol. The reactants are typically stirred together at reflux temperatures for a period of time ranging from 30 minutes to 5 hours. The resulting 1-alkylated-4-piperidone oxime or 1-alkylated-3-pyrrolidone oxime of structure 13 is recovered from the reaction zone by extractive methods as are known in the art.
In step c, the oxime functionality of the appropriate 1-alkylated-4-piperidone oxime or 1-alkylated-3-pyrrolidone oxime of structure 13 is reduced to give the corresponding 4-amino-1-piperidine and 3-amino-1-pyrrolidine derivatives (structure 4xe2x80x2).
For example, the 1-alkylated-4-piperidone oxime or 1-alkylated-3-pyrrolidone oxime of structure 13 is reacted with a suitable reducing agent, such as lithium aluminum hydride, in a suitable anhydrous solvent, such as tetrahydrofuran under an inert atmosphere. The reactants are typically stirred together at reflux temperature for a period of time ranging from 30 minutes to 5 hours. The resulting 4-amino-1-piperidine and 3-amino-1-pyrrolidine derivatives (structure 4xe2x80x2) is recovered from the reaction zone by extractive methods as are known in the art.
The following examples present typical syntheses as described in Scheme A. These examples are understood to be illustrative only and are not intended to limit the scope of the present invention in any way. As used herein, the following terms have the indicated meanings: xe2x80x9cgxe2x80x9d refers to grams; xe2x80x9cmmolxe2x80x9d refers to millimoles; xe2x80x9cmLxe2x80x9d refers to milliliters; xe2x80x9cbpxe2x80x9d refers to boiling point; xe2x80x9cxc2x0 C.xe2x80x9d refers to degrees Celsius; xe2x80x9cmm Hgxe2x80x9d refers to millimeters of mercury; xe2x80x9cxcexcLxe2x80x9d refers to microliters; xe2x80x9cxcexcgxe2x80x9d refers to micrograms; xe2x80x9cxcexcMxe2x80x9d refers to micromolar, and xe2x80x9cAPCIxe2x80x9d refers to Atmospheric Pressure Chemical Ionization. Rf values are determined by an AQ 4xc3x9750 column (YMC) with a linear gradient from 100% C to 100% D in four minutes with a two minute hold at 100% D, where C is 5:95 acetonitrile:water with 0.1% TFA, and D is 95:5 acetonitrile:water with 0.085% TFA. Molecular ion determinations were made using a Finnigan MAT SSQ-7:10 mass spectrometer.